In plasmonics and enhanced electromagnetic fields there are two main sources of electromagnetic enhancement: (1) the laser electromagnetic field is enhanced due to the addition of a field caused by the polarization of the metal particle; (2) in addition to the enhancement of the excitation laser field, there is another enhancement due to the molecule radiating an amplified emission (luminescence, Raman, etc.) field, which further polarizes the metal particle, thereby acting as an antenna to further amplify a Raman/Luminescence signal.
Electromagnetic enhancements are divided into two main classes: a) enhancements that occur only in the presence of a radiation field, and b) enhancements that can occur even in the absence of a radiation field. The first class of enhancements is further divided into several processes. Plasma resonances on the substrate surfaces, also called surface plasmons, provide a major contribution to electromagnetic enhancement. An effective type of plasmonics-active substrate consists of nanostructured metal particles, protrusions, or rough surfaces of metallic materials. Incident light irradiating these surfaces excites conduction electrons in the metal, and induces excitation of surface plasmons leading to Raman/Luminescence enhancement. At the plasmon frequency, the metal nanoparticles (or nanostructured roughness) become polarized, resulting in large field-induced polarizations and thus large local fields on the surface. These local fields increase the Luminescence/Raman emission intensity, which is proportional to the square of the applied field at the molecule. As a result, the effective electromagnetic field experienced by the analyte molecule on theses surfaces is much larger than the actual applied field. This field decreases as 1/r3 away from the surface. Therefore, in the electromagnetic models, the luminescence/Raman-active analyte molecule is not required to be in contact with the metallic surface but can be located anywhere within the range of the enhanced local field, which can polarize this molecule. The dipole oscillating at the wavelength A of Raman or luminescence can, in turn, polarize the metallic nanostructures and, if A is in resonance with the localized surface plasmons, the nanostructures can enhance the observed emission light (Raman or luminescence).
Plasmonics-active metal nanoparticles also exhibit strongly enhanced visible and near-infrared light absorption, several orders of magnitude more intense compared to conventional laser phototherapy agents. The use of plasmonic nanoparticles as highly enhanced photoabsorbing agents has thus introduced a much more selective and efficient phototherapy strategy.
One of several phenomena that can enhance the efficiency of light emitted (Raman or luminescence) from molecules adsorbed on or near a metal nanostructure is Raman scatter known as the surface enhanced Raman scattering (SERS) effect. The use of SERS measurement for a variety of chemicals including several homocyclic and heterocyclic polyaromatic compounds has been reported. [T. Vo-Dinh, M. Y. K. Hiromoto, G. M. Begun and R. L. Moody, “Surface-enhanced Raman spectroscopy for trace organic analysis,” Anal. Chem., vol. 56, 1667, 1984]. Extensive research has been devoted to understanding and modeling the Raman enhancement in SERS since the mid 1980's. For example, Kerker published models of electromagnetic field enhancements for spherical silver nanoparticles and metallic nanoshells around dielectric cores as far back as 1984 [M. M. Kerker, Acc. Chem. Res., 17, 370 (1984)]. Kerker's work illustrated theoretical calculations of electromagnetic enhancements for isolated spherical nanospheres and nanoshells at different excitation wavelengths. In his calculations, the intensity of the normally weak Raman scattering process was increased by factors as large as 1013 or 1015 for compounds adsorbed onto a SERS substrate, allowing for single-molecule detection. As a result of the electromagnetic field enhancements produced near nanostructured metal surfaces, nanoparticles have found increased use as fluorescence and Raman nanoprobes.
The theoretical models indicate that it is possible to tune the size of the nanoparticles and the nanoshells to the excitation wavelength. Experimental evidence suggests that the origin of the 106- to 1015-fold Raman enhancement primarily arises from two mechanisms: a) an electromagnetic “lightning rod” effect occurring near metal surface structures associated with large local fields caused by electromagnetic resonances, often referred to as “surface plasmons”; and b) a chemical effect associated with direct energy transfer between the molecule and the metal surface.
According to classical electromagnetic theory, electromagnetic fields can be locally amplified when light is incident on metal nanostructures. These field enhancements can be quite large (typically 106- to 107-fold, but up to 1015-fold enhancement at “hot spots”). When a nanostructured metallic surface is irradiated by an electromagnetic field (e.g., a laser beam), electrons within the conduction band begin to oscillate at a frequency equal to that of the incident light. These oscillating electrons, called “surface plasmons,” produce a secondary electric field which adds to the incident field. If these oscillating electrons are spatially confined, as is the case for isolated metallic nanospheres or roughened metallic surfaces (nanostructures), there is a characteristic frequency (the plasmon frequency) at which there is a resonant response of the collective oscillations to the incident field. This condition yields intense localized field enhancements that can interact with molecules on or near the metal surface. In an effect analogous to a “lightning rod,” secondary fields are typically most concentrated at points of high curvature on the roughened metal surface. It has been widely accepted that the electromagnetic (EM) enhancement contributes the main part of enormous enhancement factor which greatly increases the intrinsically weak normal Raman scattering cross-section. Theoretical studies of EM effects have shown that the enhanced EM fields are confined within only a tiny region near the surface of the particles, and the SERS enhancement (G) falls off as G=[r/(r+d)]12 for a single molecule located a distance d from the surface of a metal nanoparticle of radius r [K. Kneipp, H. Kneipp, I. Itzkan, R. R Dasar, M. S. Feld, J. phys. Condens. Matter 14, R597 (2002)]. Thus, the EM enhancement factor G strongly decreases with increased distance between the analyte and metal surface.
A label-free detection system that uses a SERS-based “Molecular Sentinel” (MS) probe for multiplexed detection of gene targets has been published [T. Vo-Dinh, “SERS Molecular Probe for Diagnostics and Therapy and Methods of Use Thereof”, U.S. Pat. No. 7,951,535 (2011)]. The MS nanoprobe is composed of a DNA hairpin probe (30-45 nucleotides) and metal nanoparticles. One end of the hairpin is tagged with a SERS-active label. At the other end, the probe is modified with a thiol group to covalently bond with the nanoparticle. The sequence within the loop region is complementary to the specific sequence being targeted for detection. In the absence of the target, the Raman label is in close proximity to the metal surface (closed state), and a strong SERS signal is detected due to the ‘plasmonic’ enhancement mechanism near the metallic nanoparticle. The SERS enhancement (G) falls off as G=[r/(r+d)]12 for a single analyte molecule located a distance d from the surface of a metal nanoparticle of radius r. The electromagnetic SERS enhancement strongly decreases with increased distance, due to a total intensity decay of (1/d)12. In the presence of the specific DNA target, hybridization disrupts the stem-loop configuration (open state) and separates the Raman label from the metal nanoparticle. The SERS signal is therefore significantly quenched.
Molecular sentinels (MS) have been used to detect single nucleotide polymorphisms (SNPs) in a multiplex fashion. Specifically, the MS plasmonic nanoprobe method has be used to perform multiplex detection of invasive breast cancer markers in a homogenous solution assay without washing or separation steps. This design comprised two MS nanoprobes, EBRR2-MS and KI-67-MS, to target the erbB-2 and ki-67 cancer genes, respectively. The results showed that only the SERS peaks associated with the complementary MS nanoprobes were significantly quenched when in the presence of the target DNA.
In addition to the EM enhancement contributed from individual particles, it has been observed that the EM field is particularly strong in the interstitial space between the particles. It is believed that the anomalously strong Raman signal originates from “hot spots”, i.e., regions where clusters of several closely-spaced nanoparticles are concentrated in a small volume. The high-intensity SERS then originates from the mutual enhancement of surface plasmon local electric fields of several nanoparticles that determine the dipole moment of a molecule trapped in a gap between metal surfaces. This effect is also referred to as interparticle coupling or plasmonic coupling in a network of nanoparticles (NPs), and the effect can produce a further enhancement in addition to the enhancement from individual particles. The problem of predicting the electromagnetic field in the gaps between metal nanoparticles under optical illumination has attracted interest in recent years because of the very large field enhancements induced in the particle gaps arising from surface plasmon resonances.
To investigate this feature, the electric field was calculated surrounding a finite chain of metal nanospheres or nanospheroids when illuminated with coherent light [S. J. Norton and T. Vo-Dinh, “Optical response of linear chains of nanospheres and nanospheroids,” J. Opt. Soc. Amer. 25, 2767-2775 (2007)]. The chain structure consists of nanoparticles aligned closely with small gaps between them. A method was developed applicable to spheres and spheroids which avoided the use of translational formulas at the expense of the numerical, but allowed for straightforward evaluation of certain simple integrals. In this work, the quasi-static approximation was assumed, but the basic approach could be extended to the full-wave problem, in which retardation affects were accounted for. The approach was illustrated by computing the electric field in the gaps between two spheres and between two spheroids over a range of frequencies so that the induced plasmon resonances were evident. At frequencies matching the plasmon resonances, very large field enhancements were observed to occur. It was also demonstrated how the field enhancement varied with the aspect ratio of a prolate spheroid.
Plots were generated showing the calculated values of the magnitude of the electric field between two spheres and between two prolate spheroids with two different aspect ratios. The plots showed the calculated value of the field magnitude over a range of wavelengths at a point on axis in the gap midway between the two particles. The magnitude of the incident electric field was unity; thus, the plots showed the field enhancement relative to the incident field. The observed peaks corresponded to the frequencies of the plasmon resonances. Because of the assumption of a uniform incident electric field (the quasi-static approximation), the enhancement is scale invariant; that is, the enhancement only depends on the ratio of the gap width to the particle size (e.g., the radius of a sphere or, for a spheroid, the lengths of the semi-major and semi-minor axes).
In the calculations, three pairs of particles were compared with different gaps between them: a pair of identical spheres of unit radius, and a pair of prolate spheroids with two different aspect ratios but equal in volume to that of the sphere. It was noted that the plasmon resonance red-shifted with increasing aspect ratio. In addition, for a given gap width, the two spheroids produced a noticeably larger enhancement than the two spheres. This was expected, since the smaller curvature at the spheroid ends creates a larger surface charge density and a larger field. The increased field that was observed at the ends was attributed to the “lighting rod effect.” The pair of nanospheres having an aspect ratio of 4 and a 2% gap showed an electric field enhancement in the gap of over 700 at the peak of the plasmon resonance. The total SERS signal was approximately proportional to the fourth power of the electric field magnitude, giving a total SERS enhancement of over 4×1010. However, a spatially averaged enhancement would be much less than this observed peak value. [Ref: S. J. Norton and T. Vo-Dinh, “Optical response of linear chains of nanospheres and nanospheroids,” J. Opt. Soc. Amer. 25, 2767-2775 (2007)].
The detection of nucleic acid (DNA or RNA) sequences is critical for many applications ranging from clinical diagnostics, environmental monitoring, food safety inspection, to homeland security. For medical applications nucleic acid biomarkers, such as DNA, mRNA and microRNA, have long been considered as valuable diagnostic indicators to monitor the presence of diseases and their progression. These biomarkers have great potential for early diagnosis and as therapeutic targets for effective treatment of diseases. Therefore, much effort has been devoted to the development of sensitive, selective and practical techniques for the detection of nucleic acid biomarkers.
There has been increasing interest in the use of surface-enhanced Raman scattering (SERS) for detection of nucleic acid sequences of interest (e.g. nucleic acid sequences associated with a given disease). The SERS effect greatly increases the Raman scattering cross-section enabling the use of SERS for extremely sensitive detection of the analytes. The enhancement mechanism for SERS mainly comes from intense localized electromagnetic (EM) fields arising from surface plasmon resonance in metallic nanostructures with sizes on the order of tens of nanometers. Reports on the large SERS enhancement factors of 1012-1015 have inspired the development of new sensing materials allowing sensitive detection of analytes, even down to single molecules. Together with the narrow linewidth and the molecular specific vibrational bands, SERS has now been considered as a powerful spectroscopy approach for biochemical analysis and medical diagnostics.
With recent advances in nanotechnology, a variety of different approaches have been developed to detect DNA or RNA molecules using SERS-active metallic (e.g. silver and gold) nanoparticles or nanostructured substrates. A variety of SERS plasmonic platforms have been developed for chemical and biological sensing, including a label-free detection system that uses SERS-based “Molecular Sentinel” (MS) nanoprobes for multiplexed detection of gene targets. The MS nanoprobe consists of a “stem-loop” DNA probe having a Raman label molecule at one end and a metallic nanoparticle at the other end. The detection principle of MS is based on the plasmonic enhancement mechanism near the metallic nanoparticle (i.e. the enhanced EM fields are confined near the surface of metallic nanoparticles). Upon recognition of targets, hybridization between stem-loop probes and target strands disrupts the stem-loop configuration and moves the Raman label away from the metal surface. This switches the probe conformation from a closed stem-loop structure to an open linear duplex, leading to a decrease in the SERS signal (“On-to-Off”) as the SERS enhancement strongly decreases with increased distance between the dye and metallic nanostructured surface.
New nano-plasmonic compositions having improved properties and methods of use are desirable to take advantage of the tunability of the spectral properties of the metal nanoparticles.